CN110272937B - Sensitive detection method for microorganisms in packaged drinking water - Google Patents
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- 238000011896 sensitive detection Methods 0.000 title abstract description 7
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Abstract
The invention relates to the technical field of detection of microorganisms in drinking water, in particular to a sensitive detection method of microorganisms in packaged drinking water, which is particularly suitable for detecting pseudomonas aeruginosa in packaged drinking water. According to the invention, potential microorganisms in a detection sample are effectively separated and inoculated into a liquid culture medium by a filter membrane separation method, a centrifugal separation method and the like, the detection sensitivity is increased by combining a constant-temperature shaking culture method, and the turbidity difference of a culture solution can be objectively distinguished by using an optical detection method after 8-24 hours of culture, so that the determination of a primary screening result is made, the microbial pollution risk of a product is estimated, and a production enterprise can more effectively utilize the feedback of the microbial detection result of the product to guide actual production.
Description
Technical Field
The invention relates to the technical field of detection of microorganisms in drinking water, in particular to a sensitive detection method of pseudomonas aeruginosa in packaged drinking water.
Background
Microbial contamination is an important factor affecting food safety, and is also a main cause of disqualification of packaged drinking water products, and the detection of microorganisms on production processes and finished products is a necessary means for risk control.
Pseudomonas aeruginosa is a key indicator bacterium for quality control in the production process of packaged drinking water, and the detection capability of the Pseudomonas aeruginosa is directly related to the guarantee of product quality. The detection method of the pseudomonas aeruginosa in GB 8538-2016 (inspection method for drinking Natural mineral Water) is a current national standard method for detecting the pseudomonas aeruginosa of packaging water, and the method is based on a filter membrane method, cultures in an agar culture medium mode, and identifies suspicious colonies after primary screening culture for 24-48 hours. As finished products for packaging drinking water are generally free of pollution of pseudomonas aeruginosa, colony growth cannot be detected on a primary screening culture medium, but according to the method, the primary screening result of pseudomonas aeruginosa can be judged according to the colony condition of the primary screening culture medium after 48 hours, the time is too long, and manufacturers cannot timely utilize the detection result of microorganisms to guide production.
Disclosure of Invention
Aiming at the problems that the prior detection method for packaging drinking water microorganisms such as pseudomonas aeruginosa consumes long time in preliminary screening, the detection result cannot be fed back in real time and production is guided, and the like, the invention provides the detection method for the microorganisms (especially pseudomonas aeruginosa) which has wide application range, high detection sensitivity and short preliminary screening and judging time.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A sensitive detection method for microorganisms in packaged drinking water, comprising the following steps:
s1, separating and collecting microorganisms. For the detection sample with lower pollution degree and larger sampling volume requirement, 100-1000mL of detection sample is processed by a centrifugal separation method, a filter membrane separation method and the like, so that potential microorganisms with smaller content in the detection sample and sample liquid with larger volume are separated, the attention range is reduced, potential microorganisms in the detection sample are effectively captured and collected, the influence on the representativeness of the detection sample due to limited inoculation amount is avoided, and the accuracy of the detection result is improved. For detection samples with higher pollution degree and smaller sampling volume requirement, potential microorganisms in the detection samples can be collected by direct inoculation and other modes. Meanwhile, sterile water was used as a sample, and the same method was used for treatment, as a negative control.
S2, inoculating and culturing. The collected microorganism is transferred and inoculated into 50-200mL of liquid culture medium, and is fully contacted with the culture solution. The liquid culture after inoculation was based on a 100-360rpm shaking culture apparatus, constant temperature culture. If the target microorganism is bacteria, the culture temperature is 30-42 ℃; if the target microorganism is a fungus, the cultivation temperature is 25-30 ℃. The culture conditions (culture medium, culture temperature, culture speed, culture duration, etc.) are determined by the characteristics of the target microorganism to be detected, and the optimum growth conditions of the target microorganism are selected as the culture conditions. The negative control samples were inoculated and cultured in the same manner along with the test samples.
S3, distinguishing turbidity of the culture solution. Taking out the cultured negative control sample and the detection sample, directly observing the culture solution through a light source by naked eyes, measuring the turbidity value of the culture solution by using an optical instrument, and the like, and distinguishing the turbidity difference between the negative control sample and the detection sample, thereby judging whether the detection sample has microorganism reproduction or not, and judging whether the detection sample is polluted by microorganisms or not. If it is determined that the negative control sample has no microbial proliferation, the negative control sample is effective (referred to as an effective negative control sample) and is used as a reference sample, and the turbidity difference of the culture solution between the detection sample and the negative control sample is compared. If the turbidity of the detection sample is larger than that of the effective negative control sample, judging that the detection sample is a preliminary screening suspicious sample, otherwise judging that the detection sample has no microorganism detection.
S4, colony separation, purification and identification. And (3) streaking the initially screened suspicious sample onto an agar culture medium, separating and culturing single colonies, and identifying the colonies by means of colony characteristics, microscopic examination, biochemical reaction and the like. The results of the detection of microorganisms are reported based on colony identification.
Preferably, in the step S1, the centrifugal separation method is carried out at a rotation speed of 2000-8000rpm for 2-10min at 4-30deg.C.
Preferably, in the step S1, the filter membrane used in the filter membrane separation method is a sterile filter membrane with the pore diameter less than or equal to 0.45 μm.
Preferably, the liquid medium referred to in the step S2 may be a liquid medium for promoting growth and reproduction of the target microorganism or a selective medium for inhibiting growth and reproduction of other microorganisms than the target microorganism.
Preferably, in the step S3, since a part of the liquid medium contains special substances in its components, even if no microorganism propagation exists, a certain background turbidity exists, which results in slightly poor light transmittance, and it is difficult to distinguish whether the liquid medium is contaminated by microorganisms or not only by visual observation. For the liquid culture medium with the background turbidity, an optical instrument is used for measuring the bulk turbidity values of a plurality of liquid culture mediums and calculating the average turbidity value, the background turbidity range value of the liquid culture medium is determined by establishing a background turbidity quality control table of the liquid culture medium and the like, and the turbidity value of a negative control sample is compared with the average turbidity value to judge whether the negative control sample is a valid negative control sample or not. Preferably, in step S3, if the turbidity value of the negative control sample is less than or equal to 2 times the average turbidity value, the negative control sample is an effective negative control sample.
Preferably, in the step S3, the turbidity value of the detection sample is greater than that of the 2-time effective negative control sample, and the detection sample can be judged as a preliminary screening suspicious sample.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention effectively separates potential microorganisms in the detection sample by means of a filter membrane separation method, a centrifugal separation method and the like, can avoid influencing the representativeness of the sample due to limited inoculation amount, improves the accuracy of the detection result, and is particularly suitable for packaging liquid products with lower microorganism pollution degree and larger volume such as drinking water and the like.
2. The invention combines the liquid culture medium and the shaking culture mode, can provide a richer nutrition environment and more sufficient oxygen, is favorable for providing a more proper growth environment for the growth of most of drinking water microorganisms (especially the damaged pseudomonas aeruginosa after ozone disinfection in the packaged drinking water), increases the detection sensitivity, ensures that the microorganisms, especially the pseudomonas aeruginosa, can grow faster, can judge the primary screening result after 8-24 hours, greatly shortens the culture time used for detection, and can guide the production by using the feedback detection result more efficiently for production enterprises.
3. The invention adopts the optical detection method to compare the turbidity of the detection sample with that of the negative control sample, not only can objectively, rapidly and accurately screen the sample meeting the standard, but also can avoid the influence on the result judgment caused by the uncertainty of the artificial subjective factor when the culture solution with the background turbidity is partially self and is not suitable for observing the turbidity difference with naked eyes.
In summary, for the production of packaged drinking water, as the finished product of the packaged drinking water is generally free from microbial contamination, less pseudomonas aeruginosa is polluted, and the microbial safety risk can be primarily evaluated by finishing the primary screening detection of the product. Therefore, compared with the existing detection method, the method can shorten at least more than 24 hours, obviously shorten the time required by the preliminary screening determination of the microorganism detection, improve the detection efficiency, ensure the monitoring of the food safety risk and simultaneously provide possibility for the improvement of the production efficiency of enterprises. In addition, after the primary screening detection is finished, the primary screening suspicious sample can be further separated and purified and the bacterial colony identification can be carried out, so that an accurate detection and analysis result can be obtained.
Drawings
FIG. 1 is a graph comparing CN liquid medium with microbial proliferation (positive) with CN liquid medium without microbial proliferation (negative).
Detailed Description
In order to more fully understand the technical content of the present invention, the technical scheme of the present invention is further described and illustrated below in connection with a specific experimental process.
Example 1
Sensitive detection of pseudomonas aeruginosa in packaged drinking water
And (3) filtering 250mL of water sample by using a 0.45 mu m filter membrane by adopting a filter membrane separation method, transferring the filter membrane into 50mL of CN liquid culture medium, culturing for 8-24h at a constant temperature of 36+/-1 ℃ by shaking, and setting the rotating speed to 150rpm. The water sample comprises a detection sample and sterile water used as a negative control sample.
Because the CN liquid culture medium contains substance components which enable the CN liquid culture medium to have certain background turbidity, a turbidity meter is used for measuring the bulk turbidity values of a plurality of CN liquid culture mediums and calculating the average turbidity value, a culture liquid background turbidity quality control table (for example, shown in the following table 1) of the CN liquid culture medium is established, and the background turbidity range value is determined and used for comparing the turbidity value of the negative control sample with the average turbidity value so as to judge whether the negative control sample is an effective negative control sample or not.
Taking out the cultured negative control sample and detection sample, and detecting turbidity values of the negative control sample and detection sample by naked eyes and using a turbidity meter; if the turbidity of the negative control sample shows no microorganism reproduction and accords with the value of the background turbidity range of the CN liquid culture medium (the turbidity value of the negative control sample is less than or equal to 2 times of the average turbidity value), the negative control sample is judged to be an effective negative control sample. The comparison of CN liquid medium with (positive) and without (negative) microbial proliferation is shown in figure 1.
Comparing the turbidity of the test sample with that of an effective negative control sample; if the turbidity value of the detection sample is greater than that of the 2-time effective negative control sample, judging that the detection sample is a preliminary screening suspicious sample of pseudomonas aeruginosa, otherwise, judging that the pseudomonas aeruginosa is not detected.
Purifying and identifying the initially screened suspicious sample; performing streak inoculation on the initially screened suspicious sample on a CN agar plate, and inversely placing the streak inoculated CN agar plate in a constant temperature incubator at 36+/-1 ℃ for culturing for 24 hours; if the CN agar plate shows blue or green colonies, the preliminary screening suspicious sample is judged to be detected by pseudomonas aeruginosa. If the CN agar plate does not have blue or green colonies, the CN agar plate is inspected under 360+/-20 nm ultraviolet rays, if fluorescence is observed and the CN agar plate is not blue or green, the colonies are streaked and inoculated on a nutrient agar plate and cultured for 20-24 hours at 36+/-1 ℃, and then acetamide broth confirmation test is carried out on the purified culture; if the result of the acetamide broth validation test is positive, the preliminary screening suspicious sample is judged to be detected by pseudomonas aeruginosa. If a reddish brown but non-fluorescent colony is observed, streaking the colony onto a nutrient agar plate and culturing at 36+ -1deg.C for 20-24h, and then performing an oxidase-confirming test with the purified culture; if the result of the oxidase confirmation test is positive, continuing to perform an acetamide broth confirmation test and a golden B culture medium confirmation test respectively by using the purified culture; if the results of the oxidase validation test, the acetamide broth validation test and the golden B culture medium validation test are all positive, the preliminary screening suspicious sample is judged to be detected by pseudomonas aeruginosa.
Table 1 CN liquid medium background turbidity quality control Table
The above embodiments further describe the technology of the present invention in detail by taking the detection of pseudomonas aeruginosa in packaged drinking water as an example, so as to facilitate understanding of the technology of the present invention, and the technology of the present invention is not limited to detecting pseudomonas aeruginosa in packaged drinking water.
Example 2
Sensitive detection of bacteria in packaged drinking water
And (3) filtering 250mL of water sample by using a 0.45 mu m filter membrane by adopting a filter membrane separation method, transferring the filter membrane into 50mL of TSB liquid culture medium, culturing for 8-24h at a constant temperature of 36+/-1 ℃ with shaking, and setting the rotating speed to 150rpm. The water sample comprises a detection sample and sterile water used as a negative control sample.
Taking out the negative control and the detection sample after culture, and directly observing the turbidity of the negative control and the detection sample by naked eyes. If the negative control sample is clear and not turbid and shows no microorganism reproduction, the negative control sample is judged to be an effective negative control sample.
The turbidity of the test sample was directly observed and compared with that of an effective negative control sample by naked eyes. If the turbidity of the detection sample is obviously greater than that of the effective negative control sample, judging that the detection sample is a preliminary screening suspicious sample of bacteria, otherwise judging that the bacteria are not detected.
Turbidity values of the test samples and the effective negative control samples can also be measured and compared by a nephelometer. If the turbidity value of the detection sample is greater than the turbidity value of the 2 times of the effective negative control sample, judging that the detection sample is a preliminary screening suspicious sample of bacteria, otherwise, judging that the bacteria are not detected.
In other embodiments, the detection of the potential microorganisms in the sample may also be collected by centrifugation under conditions set to: the rotation speed is 2000-8000rpm, the time is 2-10min, and the temperature is 4-30 ℃; when the potential microorganisms in the sample are collected and detected by a filter membrane separation method, other sterile filter membranes with the aperture less than or equal to 0.45 mu m can be selected as the filter membrane; in the case of shaking culture, the rotation speed is generally set to 100 to 360rpm.
The foregoing description further illustrates the technical content of the present invention by way of example only, so as to facilitate easier understanding of the reader, but is not intended to limit the embodiments of the present invention thereto, and any technical extension or recreating performed in accordance with the present invention is protected by the present invention.
Claims (5)
1. A method for detecting microorganisms in packaged drinking water by primary screening, comprising the steps of:
s1, collecting microorganisms in a detection sample; meanwhile, sterile water is used as a sample to be treated in the same way, and is used as a negative control sample;
s2, transferring and inoculating the collected microorganisms into a CN liquid culture medium, culturing the inoculated CN liquid in a vibration culture device for 24 hours at a constant temperature, and taking the optimal growth condition of the target microorganisms as a culture condition;
the negative control sample is inoculated and cultured in the same way along with the detection sample;
s3, taking out the cultured negative control sample and the detection sample, and distinguishing the turbidity difference of the culture solution between the negative control sample and the detection sample;
measuring the bulk turbidity values of a plurality of CN liquid culture media by using a turbidity meter and calculating an average turbidity value, thereby establishing a background turbidity quality control table of the CN liquid culture media; if the turbidity value of the negative control sample is less than or equal to 1.5 times of the average turbidity value of the CN liquid culture medium, the negative control sample is an effective negative control sample; comparing the turbidity difference of the culture solution between the detection sample and the effective negative control sample, and judging the detection sample as a preliminary screening suspicious sample if the turbidity value of the detection sample is greater than 1.5 times that of the effective negative control sample; otherwise, judging that the detection sample has no microorganism detection;
the microorganism is pseudomonas aeruginosa.
2. The method for detecting microorganisms in packaged drinking water according to claim 1, wherein step S3 is followed by step S4 of streaking the preliminary screening suspicious sample onto an agar medium, isolating and culturing individual colonies and identifying the colonies, and reporting the detection result of microorganisms based on the identification of the colonies.
3. The method for primary screening of microorganisms in packaged drinking water according to claim 1, wherein in step S1, 100-1000mL of the test sample is treated by centrifugation or filtration membrane separation, and potential microorganisms in the test sample are separated from the sample liquid to collect potential microorganisms, or the potential microorganisms in the test sample are collected by direct inoculation.
4. A method for the primary screening of microorganisms in packaged drinking water according to claim 3, wherein in step S1, the centrifugal separation is carried out at a rotational speed of 2000-8000rpm for a period of 2-10min at a temperature of 4-30 ℃.
5. The method for detecting microorganisms in packaged drinking water according to claim 3, wherein in the step S1, the filter membrane used in the filter membrane separation method is a sterile filter membrane having a pore size of 0.45. Mu.m.
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